In healthy women with a normal chromosome set, the Lyon hypothesis applies; this means that one of the two X chromosomes is inactivated. In rearrangements involving one of the two gonosomes, skewed X chromosome inactivation can be observed. As this can be important for the clinical course, the availability of a diagnostic in situ test for skewed X chromosome inactivation is essential. Here, we present a simple and rapid approach to distinguish between active and inactive X chromosomes based on the in vitro incorporation of 5-ethynyl-2-deoxyuridine (EdU). The prerequisite is that the person being tested has more than one X chromosome. The pattern of X chromosome inactivation is visualized at the single-cell level using EdU instead of the previously widely used 5-bromo-2′-deoxyuridine (BUdR). The fluorochrome-labeled nucleoside analog EdU is incorporated in vitro into living blood cells in late-replicating chromosome regions and can therefore also be used to specifically highlight the inactive X chromosome in a cytogenetic preparation. The EdU-based test for assessing X chromosome inactivation can only be used effectively if the X chromosomes of the index patient can be (molecular) cytogenetically distinguished from normal chromosomes under the microscope.